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International Journal of Pharmacy and Pharmaceutical Sciences Academic Sciences Vol 5, Issue 4, 2013 Research Article
EVALUATION OF POTENTIAL APHRODISIAC ACTIVITY OF MORINGA OLEIFERA SEED IN MALE
ALBINO RATS
VARSHA ZADE*, DINESH DABHADKAR, VAIBHAO THAKARE AND SHITAL PARE
Department of Zoology, Government Vidarbha Institute of Science and Humanities, Amravati 444604, Maharashtra, India.
Received: 19 Aug 2013, Revised and Accepted: 18 Sep 2013
ABSTRACT
Objective: Evaluation of the effect of the aqueous, alcohol and chloroform extract of Moringa oliefera on sexual behaviour of male albino rats. Methods: Plant extracts (aqueous, alcohol and chloroform) at doses of 100, 200 and 500 mg/kg were administrated for 21 days. The female rats involved in mating were made receptive by hormonal treatment. The general mating behaviour, libido along with orientation behaviour was studied. The effect of the extract on body weight, reproductive and vital organ weight were determined. The most effective aqueous extract was further studied for its effect on hormonal assay and compared with the standard reference drug sildenafil citrate. Similarly adverse effects and acute toxicity of the extract were also evaluated. Results: Oral administration of aqueous, alcohol and chloroform extract at doses of 100, 200 and 400 mg/kg significantly increased the Mounting Frequency, Intromission Frequency and Ejaculation latency with reduction in Mounting Latency, Intromission Latency and Post Ejaculatory Interval. It also significantly increased the libido. The extract was also observed to be devoid of any adverse effects and showed negative results for acute toxicity. Conclusion: The results of the present study demonstrate that aqueous, alcohol and chloroform extract of M. oliefera seed enhance sexual behaviour in male rats. It also thus provides a rationale for the traditional use of M. oliefera as acclaimed aphrodisiac and for the management of male sexual disorders. Keywords: Aphrodisiac, Herbal medicine, Male sexual behaviour, Male rat, Moringa oliefera, Seed
But to the best of our knowledge, there is no information in the open scientific literature that has substantiated or refuted the aphrodisiac An aphrodisiac is defined as an agent that arouses sexual desire. claims of Moringa oleifera seeds in the folklore medicine. Hence then, the Many natural substances have historically been known as present work was undertaken to validate scientifically the aphrodisiac aphrodisiac [1]. Sexual dysfunction is a repeated inability to achieve role of Moringa oleifera seeds as acclaimed by the traditional tribal user normal sexual intercourse, which includes various forms like of Melghat region of Amravati district, Maharashtra. premature ejaculation, retrograded, or retarded ejaculation, erectile dysfunction, arousal difficulties, etc. Several management options MATERIALS AND METHODS
employed are associated with some serious side effects and are not Collection of Plant Material
readily available and expensive. The search for natural supplement from medicinal plants is being intensified, probably because of The seeds Moringa oleifera plant were collected from Melghat region reduced side effect, its ready availability and reduced cost. of Amravati district during the flowering period of September to Therefore, the increasing used for search and screening of medicinal February, identified and authenticated by experts from Botanical plants with aphrodisiac potential in male has been necessitated [2]. Survey of India, Pune (Accession No. VZ- 1). Moringa oleifera (Linn) is a medicinally important plant, belonging Procurement and Rearing of Experimental Animal
to family Moringaceae. The plant is also well recognized in India, Pakistan, Bangladesh and Afghanistan as a folkloric medicine [3]. Healthy wistar strain male albino rats, two months old and weighing Moringa oleifera is a small or medium sized tree up to 10 m tall, with 200- 300 g were procured from Sudhakarrao Naik Institute of Pharmacy, thick, soft, corky, deeply fissured bark, growing mainly in semiarid, Pusad (Maharashtra). The rats were housed in polypropylene cages and tropical and subtropical areas. Different parts of the tree have been maintained under environmentally controlled room provided with a used in the traditional system of medicine. Survey in the tribal belt 12:12 hours light and dark cycle approximately at 25 C. They were fed of Melghat region (20° 51′ to 21° 46′ N and to 76° 38′ to 77° 33′ E) of on pellets (Trimurti Lab Feeds, Nagpur) and tap water ad libitum. The Amravati district of Maharashtra state of India revealed that rats were allowed to acclimatize to laboratory environment for 15 days Moringa oleifera seeds is being used traditionally as an aphrodisiac before experimentation. [4]. The seeds have been used in indigenous medicine for over many All experimental protocols were subjected to the scrutinization and decades as traditional medicine. The seeds are also known to exert approval of Institutional Animal Ethics Committee [registration its protective effect by decreasing liver lipid peroxides and, as an number 1060/ac/07/ CPCSEA (IAEC/1/2012)]. antimicrobial agent [5]. The leaves of Moringa oleifera are used as purgative, are applied as poultice to sores, rubbed on the temples for Preparation of Extract
headaches, used for piles, fevers, sore throat, bronchitis, eye and ear infections, scurvy and cataract; leaf juice is also believed to control The seeds of Moringa oleifera were collected, shade dried, powdered and glucose levels and applied to reduce glandular swelling [6, 7, 8]. The subjected to soxhlet extraction successively with distilled water, ethanol stem bark is used as an abortifacient and as an antioxidant activity and chloroform. The extract was evaporated to near dryness on a water [9, 10]. The root of Moringa oleifera were shown to possess bath, weighed and kept at 4 °C in refrigerator until further use. Phytochemical Screening
inflammatory, stimulant in paralytic afflictions; act as a cardiac/circulatory tonic, used as a laxative, abortifacient, in The presences of various constituents in the seed extract of M. treatment of rheumatism, inflammations, articular pains, lower back oleifera were determined by preliminary phytochemical screening or kidney pain and constipation [11, 7]. as per Thimmaiah [12]. Zade et al.
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Acute Toxicity Study
Copulatory Efficiency= (number of intromission/ number of mounts)× 100; Intercopulatory Efficiency= (average time between Healthy male albino rats were starved for 3- 4 hours and subjected intromissions) [18]. to acute toxicity studies as per Organization of Economic Co-operation and Development (OECD) guidelines No: 423 [13]. They Test for Libido
were divided into 4 groups of 6 animals each and kept singly in separate cages during the experiment. Group 1 represented the The test was carried out by the method of Davidson [19] modified control group, which received 10 ml/kg of distilled water orally. by Amin et al [16]. Healthy and sexually experienced male albino Groups 2- 4 received suspension of different extract (aqueous, rats (200– 300 g) that were showing brisk sexual activity were alcohol and chloroform) of Moringa oleifera seed, orally at the doses selected for the study. They were divided into 11 groups of 6 of 1000, 2000 and 5000 mg/kg daily for 7 days respectively. The animals each and kept singly in separate cages during the rats were observed continuously for 2 hours for behavioural, experiment. Group 1 represented the control group, which neurological and autonomic profile, and for next 24 and 72 hours for received 10 ml/kg of distilled water orally. Groups 2– 4 received any lethality or death. suspension of the different extract (aqueous, alcohol and chloroform) of Moringa oleifera seed orally at the doses of 100, Mating Behaviour Test
200 and 500 mg/kg, respectively, daily for 21 days at 18:00 hour. Group 5 served as standard and was given suspension of sildenafil The test was carried out by the methods of Dewsbury and Davis Jr citrate orally at the dose of 5 mg/kg, 1 hour prior to the [14] and Szechtman et al [15] modified by Amin et al [16]. Healthy commencement of the experiment. The female rats were made and sexually experienced male albino rats (200– 300 g) that were receptive by hormonal treatment and all the animals were showing brisk sexual activity were selected for the study. They accustomed to the testing condition as previously mentioned in were divided into 5 groups of 6 animals each and kept singly in mating behaviour test. The animals were observed for Mounting separate cages during the experiment. Group 1 represented the Frequency (MF) on the evening of 21st day at 20:00 hour. The control group, which received 10 ml/kg of distilled water orally. penis was exposed by retracting the sheath and 5% xylocaine Groups 2– 4 received suspension of different extract (aqueous, ointment (Lidocaine ointment, AstraZeneca Pharma) was applied alcohol and chloroform) of Moringa oleifera seed orally at the 30, 15 and 5 min before starting the observations. Each animal doses of 100, 200 and 500 mg/kg, respectively, daily for 21 days at male was placed individually in a cage and the receptive female rat 18:00 hour. Group 5 served as standard and was given suspension was introduced in the same cage. The number of mountings, of sildenafil citrate (Vigora tablets, German Remedies) orally at the intromission and ejaculation were noted. dose of 5 mg/kg, 1 hour prior to the commencement of the experiment. Since the male animals should not be tested in Orientation Activity
unfamiliar circumstances hence the animals were brought to the laboratory and exposed to dim light at the stipulated time of The test was carried out by the method of Sharma et al [20], testing daily for 6 days before the experiment. The female animals modified by Islam et al [21]. Healthy and sexually experienced male were artificially brought into oestrus (heat) [17] by the Szechtman albino rats (200– 300 g) that were showing brisk sexual activity et al [15] method (as the female rats allow mating only during the were selected for the study. They were divided into 5 groups of 6 estrus phase) They were administered suspension of ethinyl animals each and kept singly in separate cages during the oestradiol (Lynoral tablets, Organon Pharma) orally at the dose of experiment. Group 1 represented the control group, which received 100 μg/animal, 48 hour prior to the pairing plus progesterone 10 ml/kg of distilled water orally. Groups 2– 4 received suspension (Dubaget tablets, Glenmark Pharma) injected subcutaneously, at of the different extract (aqueous, alcohol and chloroform) of the dose of 1 mg/animal, 6 hour before the experiment. The Moringa oleifera seed orally at the doses of 100, 200 and 500 mg/kg, receptivity of the female animals was confirmed before the test by respectively, daily for 21 days at 18:00 hour. Group 5 served as exposing them to male animals, other than the control, standard and was given suspension of sildenafil citrate orally at the experimental and standard animals. The most receptive females dose of 5 mg/kg, 1 hour prior to the commencement of the were selected for the study. The experiment was carried out on the experiment. The orientation activity was carried out on the 21st day 21st day after commencement of the treatment of the male of treatment and was analyzed in three segments with little animals. The experiment was conducted at 20:00 hour in the same modification [21]. laboratory and under the light of same intensity. The receptive Orientation behaviour of male rats was determined using following female animals were introduced into the cages of male animals method of scoring: with 1 female to 1 male ratio. The observation for mating behaviour was immediately commenced and continued for first 2 Orientation towards female – (1 for every sniffing and 2 for every mating series. The test was terminated if the male failed to evince sexual interest. If the female did not show receptivity she was replaced by another artificially warmed female. The occurrence of Orientation towards self – (1 for every non-genital grooming and 2 events and phases of mating were recorded on audio video- for every genital grooming) cassette (Sony Handycam) as soon as they appeared. Their Orientation towards environment – (1 for every exploration, 2 for disappearance was also recorded. Later, the frequencies and every rearing and 3 for every climbing) sexual behaviour phases were determined from cassette transcriptions: number of mounts before ejaculation or Mounting The cumulative score for each orientation behaviour noted in the Frequency (MF), number of intromission before ejaculation or half hour observation period was later calculated. Intromission Frequency (IF), time from the introduction of female into the cage of the male up to the first mount or Mounting Effect on Sexual and Vital Organ Weight
Latency (ML), time from the introduction of the female up to the After the mating behaviour analysis, the next morning (Day 22), all first intromission by the male or Intromission Latency (IL), time the control, standard and experimental groups of male rats were from the first intromission of a series up to the ejaculation or evaluated for their body weight. The animals were completely Ejaculatory Latency (EL) and time from ejaculation and the first anaesthetized with anesthetic ether (Narsons Pharma), sacrificed by intromission of the following series or Post-ejaculatory interval. cervical decapacitation and then testis, seminal vesicles, epididymis, Using the above parameters of sexual behaviour, the following vas-deference, penis and prostate glands along with vital organ like computed parameters were calculated: % index libido= (number liver, kidney, adrenal gland, and spleen were carefully removed and mated/ number paired)×100; % Mounted= (number mounted/ weighed using digital electronic balance [22, 23, 24]. number paired)× 100; % Intromitted= (number of rats that Statistical Methods
intromitted/ number paired)× 100, Intromission ratio= (number of intromission/ number of mount + number of intromission), % All the data are expressed as mean ± S.E. Statistical analysis was Ejaculated= (number of rats that ejaculated/ number paired) × 100; done by Student's t-test and one way ANOVA [25]. Zade et al.
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mg/kg body weight significantly increased the Mounting Frequency (MF) (P<0.001), Intromission Frequency (IF) (P<0.001) and Phytochemical Screening
Ejaculatory Latency (EL) (P<0.001). Similarly it also causes Preliminary phytochemical screening of the seed extract of Moringa significant reduction in the Mounting Latency (ML) (P<0.001) and oleifera revealed the presence of alkaloids, flavonoids, steroids, Intromission Latency (IL) (P<0.001) in experimental animals as phenolics, tannins and saponines whereas anthraquinones were not compared to control group. Similarly, the standard drug also increased the MF, IF and EL as well as decreased the ML (P<0.001) and IL (P<0.001) in a highly significant manner as compared to Acute Toxicity Study
control animals. The most appreciable effect was observed in the aqueous extract of M. oleifara at the dose of 100, 200 and 500 mg/kg Clinical toxicity symptoms such as respiratory distress, salivation, weight body weight (Table- 1). The alteration in these parameters was loss and change in appearance of hair as well as maternal mortality were statistically significant. not observed at any period of the experiment. Similarly no mortality and changes in the behavioural, neurological and autonomic profile were The computed male sexual behaviour parameters which include observed in treated groups of the rats up to highest dose of 5000 mg/kg percentage index of libido, % mounted, % intromitted, % ejaculated body weight. Hence one tenth of treated dose (500 mg/kg b. w.) was and copulatory efficiency were found to be higher in the extract selected for present investigation. treated animals compared to the distilled water treated control animals (Table- 2). In contrast, the aqueous, alcohol and chloroform Effect of the Extract on Mating Behaviour
seed extract of Moringa oleifera reduced the intercopulatory interval The administration of Moringa oleifera aqueous, chloroform and of the animals in dose related manner compared to the distilled alcohol seed extract for 21 days to male rats resulted in remarkable water administered control animals. Similarly the standard drug increase in the sexual vigor of the male rats, as evidenced by the (Sildenafil citrate) treated group of animals also exhibited a different sexual behaviour parameters studied. The results of mating decrease in the intercopulatory interval when compared to control behaviour test show that the seed extract of Moringa oleifera animals (P<0.001). The decrease observed was statistically (aqueous, alcohol and chloroform) at the dose of 100, 200 and 500 significant (P < 0.001, P < 0.01 and P < 0.05). Table 1: Effect of aqueous, alcohol and chloroform extract of Moringa oleifera seed on mating behaviour in male rats
Treatment
Parameters
Mount Latency
Ejaculation
Ejaculation
Frequency
Latency (in Sec)
Frequency
Latency (in
Group- I Control
Group- II
Aqueous extract
Group- III
Alcoholic extract
Group- IV
Chloroform
Sildenafil citrate
Values in Mean± S.E. (Standard error), n=6, *P<0.05, **P<0.01, ***P<0.001, when compared with control, ns- non significant. Table 2: Effect of aqueous, alcohol and chloroform extract of Moringa oleifera seed on computed male rat sexual behaviour parameters
Treatment
Parameters
Intromission %Ejaculated
of libido
Intromitted
Copularory
interval (in sec)
Efficiency
Control
Group- II
Group- III
Alcoholic
Group- IV
Chloroform
Sildenafil
citrate
Values in Mean± S.E. (Standard error), n=6, *P<0.05, **P<0.01, ***P<0.001, when compared with control, ns- non significant. Zade et al.
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Effect of the Extract on Libido
frequency increases in a significant manner in all the extract treated group in a dose dependent manner, however ejaculation The results obtained in the test for libido shows that the aqueous, was found to be absent in100 mg/kg b. w. alcohol extract and in alcohol and chloroform seed extract of Moringa oleifera at the dose 100 and 200 mg/kg b. w. chloroform extract treated groups. of 100, 200 and 500 mg/kg, significantly increased the Mounting However a strikingly increased libido activity was observed in the Frequency (MF) (P < 0.001, P < 0.01 and P < 0.05) as compared to 500 mg/kg body weight treated animals of all the extract treated control group. The standard drug also significantly increased the groups with a marked increase in aqueous extract treated group MF (P < 0.001) as compared to control animals. The intromission Table 3: Effect of aqueous, alcohol and chloroform extract of Moringa oleifera seed on mounting frequency (test for libido) in male rats
Treatment Groups
Parameters
Doses
Mounting Frequency
Ejaculation (EJ)
(mg/kg body wt.)
Frequency (IF)
Group-III
Aqueous extract
Group-IV
Alcoholic extract
Chloroform extract
Group-II
Sildenafil citrate
Values in Mean± S.E. (Standard error), n=6, *P<0.05, **P<0.01, ***P<0.001, when compared with control, ns- non significant Effect of the Extract on Orientation Behaviour
towards environment (exploration, raring and climbing) was significantly decreased in experimental animals and moderately The aqueous, alcohol and chloroform extracts Moringa oleifera seed decreased in standard group. The studies on the genital grooming of at the dose level of 100, 200 and 500 mg/kg body weight markedly male rats revealed that there was significant increase in genital influenced the orientation behaviour of the treated animals, which grooming (P < 0.001, P < 0.01 and P < 0.05) in all extract treated showed more attraction towards female rats. The studies revealed groups, while moderate decrease in non-genital grooming was significant increase in number of licking (P< 0.001, P< 0.01) and in observed as compared with the control group. The standard drug the anogenital smelling (P < 0.001, P < 0.01 and P < 0.05) of treated also shows significant increase in genital grooming and decrease male rats towards receptive female comparable to the standard drug non-genital grooming of male rats as compared to control group treated group of animals. The behavioural assessment of rats Table 4: Effect of aqueous, alcohol and chloroform extract of Moringa oleifera seed on orientation activity in male rats
Treatment
Mean activity score towards
Mean activity score towards Environment
Mean activity Score towards
Anogenital
Exploration
Climbing
Nongenital
smelling
grooming
grooming
Control
Group- II
Group-III
Alcoholic
Group- IV
Chloroform
Sildenafil
citrate
Values in Mean± S.E. (Standard error), n=6, *P<0.05, **P<0.01, ***P<0.001, when compared with control, ns- non significant. Effect of Extract on Sexual and Vital Organ Weight
seminal vesicle, penis and vas- deferens increased significantly when compared to control (P < 0.001, P < 0.01 and P < 0.05). Similarly, there The intragastric (i. g.) administration of aqueous, alcohol and chloroform was significant increase in the relative weight of the vital organs like seed extract of Moringa oleifera at the dose of 100, 200 and 500 mg/kg, liver, kidney, adrenal gland and spleen (P < 0.001, P < 0.01 and P < 0.05), caused an increase in body weight, significantly, when initial and final when compared with control animal group (Table- 5). The significant body weight were compared. The relative weight of the reproductive increase in the weight of reproductive and vital organs was also organ like testes, caput segment of the epididymis, ventral prostate, observed in standard group when compared to control. Zade et al.
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Table 5: Effect of aqueous, alcohol and chloroform extract of Moringa oleifera seed on body weight, male reproductive organ and vital
organ weights of male rats
Body weight (gm)
Group-III
Group-IV
Alcoholic
Group-II
l citrate
Values in Mean± S.E. (Standard error), n=6, *P<0.05, **P<0.01, ***P<0.001, when compared with control, ns- non significant. DISCUSSION
therefore boost the level of testosterone in the body as well as trigger libido enhancing effect [29] observed in this study. The The seed of Moringa oleifera is been used traditionally in use by the presence of flavonoids in the Moringa oleifera extract which has tribals of Melghat region as a means of treating sexual inadequacy been implicated to have a role in altering androgen levels [30] may and stimulating sexual vigor even without recourse to the scientific also be responsible for the enhanced male sexual behaviour in this validity of the claim. Hence this study was carried out to validate study. The alkaloid is also reported to cause facilitation of sexual scientifically this tribal claim. behaviour and has effect on sexual behaviour [31]. Thus the improvement in sexual function demonstrated in the current study The phytochemical screening helps to reveal the chemical might thus be due to the presence of compounds such as flavonoids, constituent of the plant extract and the one that predominates over saponins and alkaloids in Moringa oleifera seed extract. Further the other. It may also be used to search for bioactive agents as study is required to identify the active constituents responsible for starting products used in the partial synthesis of some useful drugs the sexual function improvement activities. [26]. The preliminary phytochemical screenings of the seed extract of Moringa oleifera revealed the presence of alkaloids, flavonoids, In the present study, clinical toxicity symptoms such as respiratory steroids, phenolics, tannins. It has been reported that steroids and distress, salivation, weight loss and change in appearance of hair as saponin constituents found in many plants possess fertility well as maternal mortality were not observed at any period of the potentiating properties, and are useful in the treatment of experiment. Hence it can be suggested that short term use of M. impotence [27]. Saponins found primarily in the leaf of Tribulis oleifera seed extract for this purpose is apparently safe. Similar terrestris L. have been used as an aphrodisiac agent in both; Indian finding was also observed by Tajuddin, et al [32], while working on and Chinese traditional system of medicine [28]. The saponins may ethanolic extract of Myristica fragrans. Zade et al.
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In male rats, latency for mount and intromission are considered as been shown to improve sexual function and libido [39], in addition indicators of the sexual motivation, whereas intromission and to the intensity of orgasm and ejaculations which might also be ejaculation frequencies are considered as behavioural indication of expected to improve [40]. Similar conclusion was recorded by sexual performance and facilitation [33]. After treatment with the Watcho et al [41], while working on hexane extract of Mondia whitei various experimental doses of seed extract of Moringa oleifera there on the reproductive organ of male rats. was a significant decrease in the latency for mount and intromission latencies indicating enhancing of sexual motivation, which was CONCLUSION
predominant at 21st day of observation. Similarly an increase in the This aphrodisiac activity study lends support to the claim for number of ejaculations with an increase in the ejaculation latency traditional usage of Moringa oleifera as a sexual function enhancing indicated an increase in the sexual performance. The aqueous, medicine. Thus, this study may prove to be an effective and safe alcohol and chloroform extracts have a pronounced effect on sexual alternative remedy in sexual disorders. Work is in progress on the behaviour shown by significant increase in Mounting frequency isolation and characterization of the aphrodisiac principle in the (MF) and Intromission frequency (IF) as compared to control. The plant extract, the actual mechanism of action of the crude extract MF and IF are considered the indices of both libido and potency. The and bioactive agents. significant increase in Ejaculation latency (EL) suggests that the all experimental extracts and standard drug prolonged the duration of coitus, which is an indicator of increase in sexual motivation [34]. Authors are thankful to University Grant Commission Government of The significant increase in computed male sexual behaviour India for funding the present work as a part of the Post doctoral parameters like % mounted , % intromitted, % ejaculated and the program in the form of Research awards. The authors are grateful to reduction in intercopulatory efficiency are indications of sustained CPCSEA, Chennai, Ministry of Justice and Empowerment, increase in sexual activity and aphrodisiac property inherent in the Government of India and IAEC, Government Vidarbha Institute of plant extract [35]. The present findings show that the aqueous, Science and Humanities, Amravati (M.S) for giving the permission alcohol and chloroform seed extract of Moringa oleifera produces a for doing the experimental work on rat. striking enhancement of over- all sexual performance of normal animals. Our finding are corroborated with the aphrodisiac effect of REFERENCES
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